Process for producing xanthomonas hydrophilic colloid



United States Patent 3,232,929 PRUCESS FOR PRODUCING XANTHOMONASHYDRGPHILIC COLLOID William H. McNeely, 5343 W. Fall View Drive, SanDiego, Cflifi, and John J. OConnell, 4951 Art St., San Diego 15, Calif.No Drawing. Filed May 25, 1960, Ser. No. 31,510' 5 Claims. (Cl. 260-209)This invention relates to valuable improvements in methods of obtainingXanthomonas hydrophilic colloid. The invention relates particularly toan improved method of ,recovery of Xanthomonas hydrophilic colloid fromthe clarified fermentation liquor wherein the aforesaid colloid wasproduced biosynthetically.

' Itis known that Xanthomonas hydrophilic colloid can be produced bytransferring the bacterium Xamhomonas campestriato a suitable medium andconditioning it to vigorous growth through two stages before allowing itto grow in a final medium containing 3% glucose. After 96 hours at 30 C.with suitable aeration and stirring the aforementioned colloid will 'beproduced in approximately 1% concentration. The viscosity is normallyquite high so that the medium must be diluted to much lowerconcentrations in order that the insoluble materials therein containedmay be removed. The known methods of preparation have called for adilution to 0.6% colloid concentration or less, and centrifugation as ameans of clarification. To recover the colloid in solid form hasrequired precipitation with a water miscible solvent (for example,methanol) in which the colloid is insoluble. This requires very largeratios of solvent to colloid. Even with a good solvent recovery system,the minor losses to be expected make the process very costly. Theclarification also would be faster and less expensive if it werepossible to dilute the fermentation liquor to much lower concentrations(lower viscosities). However, the solvent costs would be multiplied toan impractical point were this dilution substantial.

Means other than using solvents for precipitating the colloid haveseemed non-existent. The pure colloid has excellent compatibilities withsalts of the common cations (sodium, potassium, ammonium, calcium,magnesium, aluminum, etc.). It is not precipitated by acids such ashydrochloric, sulfuric, phosphoric or acetic, nor by alkalies such assodium, potassium or ammonium hydroxide. It is, thus, surprising thatwhere neither calcium salts or alkalies cause precipitation bythemselves, by our invention the materialprecipitates with a combinationof calcium and alkali. The precipitation is relatively unchanged bydilution within the useful range. Thus, the mate-rials needed to recovera given weight of colloid from a 0.2% concentration are notsubstantially different than those needed in the case of a 0.6%concentration. Solvent requirements by the known method would be tripledat 0.2% compared to 0.6%. The costs of the materials from the alkalinecalcium precipitation are a small fraction of those by the solventprocedure.

Yet another advantage is found in our discovery. The precipitation canbe arranged so that fibers of colloid result which are particularlysuited to 'washing, extraction, dewatering or other treatments.Machinery for handling such a material in a continuous process is simplein design and relatively inexpensive.

3,232,929 Patented Feb. 1, 1 966 As the second part of our invention forthe recovery, these precipitated fibers can be recovered in difierentways to give the soluble colloid.

One method that we have found is to neutralize the fibers (pressed toabout 30% solids) with an acid, and dry the material. This producessoluble colloid containing a calcium salt. To avoid a hygroscopic natureone may choose to make the calcium salt an insoluble one (example,calcium sulfate). For some usese the product can be used directly. Forothers, if a calcium salt is chosen which has solvent solubility orpartial solubility (example, calcium acetate) the dried product can thenbe extracted with the solvent to remove the salt.

Another method is to suspend the pressed fibers in a water-misciblealcohol and neutralize the material with a suitable acid havingsolubility in the alcohol, such as hydrochloric acid. The resultingcalcium salt, e.g., calcium chloride, is then extracted with an organicsolvent in which the calcium salt is soluble.

Yet a third procedure that we have invented is to place the fibers inalcohol containing sufiicient HCl to make the mix acidic, press out thefluid, wash with more alcohol and then neutralize with a base of thecation desired to be attached to the carboxyls of the colloid.

The Xanthomonas hydrophilic colloid that is contemplated in ourinvention is a colloid prepared by the bacterium Xanthomonas campestris.This high molecular weight, exocellular material is a polymer comprisingmannose, glucose and potassium glucuronate units.

In preparing the said colloid as aforesaid, it is convenient to use cornsteep liquor or distillers dry solubles as an organic nitrogen source.It is expedient to grow the culture in two intermediate stages prior tothe final inoculation in order to encourage vigorous growth of thebacteria. These stages may be carried out in media having a pH of about7. In a first stage a transfer from an agar slant to a dilute glucosebroth may be made and the bacteria cultured for 24 hours under vigorousagitation and aeration at a temperature of about 30 C. The culture soproduced may then be used to inoculate a higher glucose (3%) contentbroth of larger volume in a second intermediate stage. In this stage thereaction may be permited to continue for 24 hours under the sameconditions as the first stage. The culture so acclimated for use withglucose by the aforementioned first and second stages is then added tothe final glucose medium. In the aforesaid method of preparingXanthomonas compestris hydrophilic colloid, a loopful of organism fromthe agar slant is adequate for the first stage comprising 200milliliters of the said glucose media. The second stage comprised thematerial resulting from the first stage together with 9 times its volumeof a 3% glucose media. In the final stage the material produced in thesecond stage was admixed with 19 times its volume of the final media.The final media contained 3% glucose, 0.5% distillers dry soluble, 0.5%dipotassium phosphate, 0.1% magnesium sulphate having 7 molecules ofwater of crystallization and water. The reaction in the final stage wascarried out for 96 hours at 30 C.-with vigorous agitation and aeration.

By the known solvent method the mixture is then diluted with an equalamount of water and methanol to give 24.7% methanol by weight in themixture. It is centrifuged twice and the methanol increased to 56%.

The precipitate is recovered, redissolved and retreated once more. Thefibers obtained the second time are hardened in approximately 5 volumesof methanol, dried and milled. In all, methanol in excess of 400 partsper part of colloid is used by this procedure, which is costly in bothmaterials and equipment. On the other hand, by proceeding in accordancewith our invention, the mixture obtained after the 96 hours fermentationis diluted with 5 volumes of water and then centrifuged. Thecentrifuging thus produces a clarified liquor much more readily. It isthen precipitated by hydrated lime. As an illustration of this procedurewe give the following:

EXAMPLE A Precipitation process To a slurry of 20 pounds of hydratedlime in 200 pounds of water add, in a thin stream, clarified fermentedliquor which contains pounds of Xanthomonas hydrophilic colloid atapproximately 0.2% concentration. Stir the mixture slowly during theaddition. Pass the slurry over a screen, and dewater the fibers whichare separated in a press.

The moisture content of these fibers varies with the means ofdewatering. We have worked with fibers having a moisture content ofapproximately 70%.

By this invention these fibers may be used in the form just obtained,or, preferably, they may be further treated. We now give three basicmethods within the broad scope of our invention for reconstituting thefibers to a watersoluble form where this is desired:

EXAMPLE I Neutralization Place the precipitated and dewatered fibersfrom Example A in a heavy duty mixer so that 10 pounds of Xanth-omonashydrophilic colloid is therein. Add sulfuric acid until the mix remainsat a neutral pH. Dry the resulting paste in thin sheets and mill it.

Such a product contains calcium sulfate, and the soluble colloid. It canbe used in applications where calcium sulfate is acceptable.

The sulfuric acid can be replaced by other acids, such as hydrochloricacid, phosphoric acid, carbonic acid or acetic acid on an equivalentbasis, and the corresponding calcium salt of the acid used is formed.

EXAMPLE II Neutralization and extraction Place the precipitated anddewatered fibers from Example A in a heavy duty mixer so that 10 poundsof Xanthomonas hydrophilic colloid is therein. Add acetic acid until thepH remains neutral. Dry the resulting paste in thin sheets and mill to apowder. Extract the powder in methanol in a Soxhlet type of extractoruntil it is essentially free of acetate salt.

The acetic acid can be replaced by other acids, such as hydrochloricacid on an equivalent basis, the alternative acid being chosen so as togive a calcium salt which is soluble in the organic solvent used toremove it, such as for example, methanol, ethanol, and the like.

EXAMPLE III Acidic solvent wash and neutralization To a heavy duty mixeradd 65 pounds of methanol and 60 pounds of 37% I-ICl, and to this addthe fibers from Example A so that 10 pounds of Xanthomonas hydrophiliccolloid is present. Mix for minutes and press out the fibers. Wash thefibers in 2 baths containing a mixture of pounds of methanol and 25pounds of water. Suspend the fibers in 25 pounds of methanol and 25pounds of water and add a 4 normal solution of potassium hydroxide untilthe fibers are neutralized. Press the liquid from the fibers. Dry thefibers and mill them.

We have found that to have the carboxyls of the colloid neutralized witha cation other than potassium, the base of the cation desired should besubstituted in place of the potassium hydroxide in Example III. Thus,sodium hydroxide, lithium hydroxide, and the like may be used, asdesired.

We have also found that the acid-washed fibers need not be suspended inmethanol for the potassium hydroxide neutralization whenever equipmentis available that can dry the resulting paste satisfactorily.

In Examples II and III we have found that the methanol can be replacedby ethanol, isopropanol or acetone.

In Example II, extractors other than the Soxhlet type can be used and insome of these it is advantageous to use some water in the methanol toincrease the salt solubility and the penetration of the powderparticles.

The water-soluble product obtained by proceeding in. accordance with ourinvention, as set forth in Examples: I, II and III, may be used for awide variety of purposes. For example, they may be used to increase thefoam in: carbonated beverages, such as beer, root beer, cream soda: andthe like. They may be used as additives to drilling: muds, both asthickening and suspending agents, and particularly as concerns thefibrous precipitate which is the primary product as obtained forexample, in Example A,. for overcoming lost circulation in drillingthrough frac-- tured strata. The products obtained in accordance with'our invention are suitable for a wide variety of ceramic uses, such asfor suspending enamel frits. They have great utility for foods, such asfor example, suspending particulate food in a gelatinous matrix, as forexample, in aspic, jellied fruit desserts, canned sea food, and thelike. They also are useful as fortifying agents for candies, jams andjellies and as thickening and smoothing agents for certain dairyproducts, such as process cheese and ice cream. They may be used as asize for paper and textiles and are even capable of being spun intotextile fibers themselves by suitable treatments.

For convenience the disclosure and the claims which follow utilize theterm hydrated lime. It will be evident to those skilled in the art thatquicklime or slaked lime may be the starting material, since as soon asquicklime is slurried in water, the quicklime becomes hydrated lime.

The relative proportions of reactants are not especially critical, andin general are dictated primarily by good plant practice. For example,if in the process set forth in Example A too little hydrated lime wereused, some precipitate would be obtained, but the remainder of theXanthomonas colloid would simply be wasted. On the other hand, it wouldbe pointless to use a great excess of hydrated lime as this would simplyload the precipitate ob tained with dead weight in the form of hydratedlime.

It will be understood, accordingly, that while exemplary proportions andstarting materials and reactants have been given in order to elucidatethe invention, the scope of the latter is broad and many permutations,substitutions, changes in temperature and like working con ditions maybe used while still proceeding in accordance with our invention, allwithin the scope of the claims which follow.

Having described our invention, we claim:

1. A process of preparing a water-soluble Xanthomonas colloid from anaqueous dispersion of a Xanthomonas colloid which comprises: preparing aslurry of hydrated lime in water; admixing said dispersion with saidslurry; recovering the fibrous precipitate which forms; admixing saidprecipitate with a solution of water-miscible organic solvent and anacid which is soluble in said organi-c solvent and whole calcium salt issoluble in said organic solvent; subsequently separating theXanthomonas; colloid so obtained from said organic solvent and said acidsolution; and thereafter neutralizing said, XEIlIhQIILQ; nas colloidwith a base.

2. The process of claim 1 wherein the acid is hydrochloric acid and thebase is chosen from the group consisting of potassium hydroxide,potassium carbonate, sodium hydroxide, sodium carbonate and ammoniumhydroxide.

3. The process of claim 1 wherein the acid is hydrochloric acid, thebase is chosen from the group consisting of potassium hydroxide,potassium carbonate, sodium hydroxide, sodium carbonate and ammoniumhydroxide, and the organic solvent is chosen from the group consistingof methanol, ethanol isopropanol, and acetone.

4. The process of claim 1 wherein the acid is hydrochloric acid, thebase is sodium hydroxide, and the Watermiscible solvent is methanol.

5. The process of claim 1 wherein the acid is hydrochloric acid, thebase is potassium hydroxide, and the Water-miscible solvent is methanol.

References Cited by the Examiner UNITED STATES PATENTS 2,685,579 8/1954-Wimmer 19531 2,686,777 8/1954 Wimrner l953'l 3,020,206 2/1962 Patton eta1 252-855 OTHER REFERENCES 15 JULIUS GREENWA'LD, Primary Examiner.

ALBERT T. MEYERS, Examiner.

1. A PROCESS OF PREPARING A WATER-SOLUBLE XANTHOMONAS COLLOID FROM ANAQUEOUS DISPERSION OF A XANTHOMONAS COLLOID WHICH COMPRISES: PREPARING ASLURRY OF HYDRATED LIME IN WATER; ADMIXING SAID DISPERSION WITH SAIDSLURRY; RECOVERING THE FIBROUS PREICPITATE WHICH FORMS; ADMIXING SAIDPRECIPITATE WITH A SOLUTION OF WATER-MISCIBLE ORGANIC SOLVENT AN AN ACIDWHICH IS SOLUBLE IN SAID ORGANIC SOLVENT AND WHOLE CALCIUM SALT ISSOLUBLE IN SAID ORGANIC SOLVENT; SUBSEQUENTLY SEPARATING THE XANTHOMONASCOLLOID SO OBTAINED FROM SAID ORGANIC SOLVENT AND SAID ACID SOLUTION;AND THEREAFTER NEUTRALIZING SAID XANTHOMONAS COLLOID WITH A BASE.